Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations

Apical membrane of renal proximal tubule cells is extremely sensitive to is chemia, with structural alterations occurring within 5 min. These changes a re felt secondary to actin cytoskeletal disruption, yet the mechanism respo nsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-bindin g protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F- actin binding, depolymerization, and severing, and because ADF activation o ccurs by dephosphorylation, we questioned whether ADF played a role in micr ovilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluo rescence and Western blot studies on cortical tissue documented the presenc e of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylate d forms were identified. During ischemia, marked alterations occurred. Intr aluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occ urring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia , total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collect ed under physiological conditions did not contain ADF or actin, whereas uri ne collected after 30 min of ischemia contained both ADF and actin. Reperfu sion was associated with normalization of cellular pADF levels, pADF intrac ellular distribution, and repair of apical microvilli. These data suggest t hat activation of ADF during ischemia via dephosphorylation is, in part, re sponsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.